Summary/Abstract The goal of Project 1 is to establish that a botanical supplement provides resilience against stress-induced pathophysiological responses that confer susceptibility to depressive behavior. This proposal will investigate how polyphenol metabolites of a bioactive dietary polyphenol preparation (BDPP) engage biomolecular and genetic targets in microglia, medium spiny neurons (MSNs), and blood brain barrier (BBB) cells to promote resilience of neuronal function and behavior in response to stress. Recent studies show that dysfunctional immune activity confers susceptibility to stress by affecting activity of these aforementioned cells. Systemic upregulation of inflammatory cytokines, in particular interleukin-6 (IL-6), by stress is observed in model systems of depression, as well as in patients with MDD. From preliminary studies involving a validated model of social stress, we show persistent IL-6 expression from leukocytes affects the BBB function only in stress- susceptible mice. We present evidence that BDPP bioavailable metabolites may promote resilience to social stress by suppressing production of cytokines, which is associated with maintenance of BBB integrity. Our first goal is therefore to establish if BDPP provides resilience against stress-induced depression by targeting mechanisms associated with BBB function. This aim will determine whether a botanical supplement prevents BBB permeability and infiltration of peripheral immune factors through the neuroendothelium, and will characterize biomolecular targets of BDPP metabolites in endothelial cells. Our preliminary studies show BDPP supplementation suppresses stress-induced microglia hyper-ramification and upregulation of inflammatory genes, which are associated with stress-susceptibility. Our second goal is to conduct an unbiased screen to identify molecular bioactivities of BDPP metabolites in microglia in response to stress. This aim involves a cell-specific RNA-sequencing technique termed translating ribosomal affinity purification (TRAP). Moreover, Aim 2 will characterize how increased activity of toll/nod-like receptors in microglia confer susceptibility to stress-induced depression, and act as proximate biological targets for BDPP metabolites. Finally, we show how BDPP metabolites prevent maladaptive glutamatergic synapse generation in the NAc in response to stress by regulating expression of synaptic genes in MSNs. We also show that peripheral IL-6 production by peripheral leukocytes in response to stress is critical for facilitating aberrant synapse formation in the NAc. The final goal will therefore be to conduct an unbiased screen to identify molecular bioactivities of BDPP metabolites in D2 MSNs using the TRAP method and to investigate if peripheral IL-6 diffusion into the brain activates microglia via the IL-6R to effect regulation of NAc synapse densities. This would provide a mechanism for why suppression of peripheral IL-6 by a botanical supplement can promote resilient behaviors for a future clinical trial. Together, Project 1 will validate that pathophysiological responses associated with IL-6 can be suppressed by a botanical supplement to promote resilience to stress.